Analysis of pfnhe expression levels in recombinant clones. (A) RT-PCR analysis with primers p8+p9, specific for the functional recombinant pfnhe allele, reveals a 0.4 kb transcript from the knockdown clones. (B) Northern hybridization of total RNA from synchronized schizont stage parasites probed with a pfnhe fragment (top panel) reveals substantially reduced steady-state pfnhe transcription in the recombinant clones compared to their parental lines. Loading controls are shown in the middle panel (ethidium bromide (EtBr) stained gel), and the bottom panel (same blot rehybridized with an ef-1α probe). (C) Western blot of total protein samples from recombinant and parental lines probed with antibodies to PfNHE or the P. falciparum Golgi marker PfERD2.

Fluorimetric pH measurement of the P. falciparum cytosol. Bright field image (A) of infected and uninfected RBCs loaded with the pH sensitive dye BCPCF-AM. A fluorescence image obtained following excitation at 495 nm is shown in (B), and panels A and B are merged in (C). The membranes of the RBC, parasite (P), and digestive vacuole (DV) are indicated. All pH measurements were made in individual parasites by examining a region of interest within the parasite cytoplasm (illustrated here as a circle). The scale bar is 10 µm. (D) BCPCF loaded parasitized erythrocytes were treated with 0.01% saponin to permeabilize the RBC membrane, and then exposed to rapidly alternating 495 nm and 440 nm excitation light. The pH within the parasite cytoplasm was derived from the ratio of the fluorescent emission derived from 495/440 nm excitation. The BCPCF probe was calibrated within each individual parasite by exchanging the perfusate with an iso-osmotic high K⁺ solution in the presence of 1 µM nigericin, over a range of pre-measured pH values (6.7, 7.0, 7.3, 7.6). (E) The average cytosolic pH values of the strains examined in this study are shown. For each of the parental strains (GC03, 3BA6, 1BB5), two pfnhe underexpressing strains were examined. The cytosolic pH value of each line (shown as the mean±SEM) represents data from 39–72 individual parasites, acquired on 3 separate days.

Generation of pfnhe recombinant knockdown clones. (A) Schematic representation of pCam_bsd/pfnheΔ3’UTR plasmid integration into the pfnhe locus via homologous recombination and single-crossover to replace the pfnhe endogenous 3’UTR with the truncated pfcrt 3’UTR. This resulted in the generation of an upstream functional recombinant pfnhe gene possessing the endogenous pfnhe promoter, the full-length coding sequence and the 150 bp truncated 3’UTR of pfcrt. The downstream integrated blasticidin S deaminase selection marker (bsd), driven by the calmodulin (cam) 5’UTR and the hrp2 3’UTR, is followed immediately by a nonfunctional pfnhe remnant. This downstream, disrupted locus lacks 4.6 kb of 5’ pfnhe coding sequence and its promoter and retains the endogenous 3’UTR. (B) PCR detection of the recombinant functional pfnhe locus with primers p6+p5 (top panel), or the endogenous undisrupted pfnhe with primers p6+p7 (bottom panel), for the pfnhe parental lines (WT) and their respective knockdown clones (listed as M-1 and M-2 for each genetic background). (C) Southern hybridization of EcoRV-digested genomic DNA from recombinant and parental lines probed with a pfnhe 3’ coding fragment.

Antimalarial susceptibility profiles of pfnhe-modified clones and their parental lines. This is shown as a graphical representation of IC₅₀ mean±SEM values for Quinine (QN)±Verapamil (VP), Chloroquine (CQ)±VP, Mefloquine (MFQ), and Lumefantrine (LMF) for the various lines. Values (listed in Table 3) were calculated from 5–9 separate assays performed in duplicate (*, P<0.05).

Nigericin/BSA pH clamp method applied to PfNHE recombinant clones and their parental lines. (A) Data were obtained using the nigericin/BSA pH clamp method that has been reported to measure NHE activity. Saponin-permeabilized iRBCs were perfused with high K⁺ containing E1 (K⁺ E1) buffer at pH 7.5. 1 µM nigericin was added to the perfusate at the downward arrow marked (N), first at pH 7.5 and then at pH 6.0. After equilibration of the cytosolic BCPCF probe signal at pH 6.0, nigericin was removed and 5 mg/ml BSA was added to the perfusate at the downward arrow marked (B) to adsorb the remaining nigericin, clamping the cytoplasmic pH at 6.0. The perfusion pH was then returned to 7.4, either in the presence of Na⁺ (solid trace) or in a Na⁺ free NMDG⁺ buffer (dashed trace). Each trace represents the average of over 10 individually analyzed parasites within one microscopic field. (B) Prior to the time zero in these traces the full pH clamp experiment was performed as in panel (A) up to 400 seconds, where the buffer pH was returned to 7.4. Data from three separate experiments with 3BA6 parasites (closed triangles) in which the return to pH 7.4 Na⁺ E1 buffer is shown on an expanded time scale (i.e., from ~400 seconds to end in panel A). Parental 3BA6 parasites exhibit significant variability in their realkalinization rate upon return to Na⁺ E1 (closed triangles), but show very little alkalinization upon return to NMDG⁺ E1 (open triangles). A 3BA6 NHE under expressing strain (closed squares) exhibits a rate of alkalinization within the range of the parental strain responses. Traces are representative of many experiments, conducted on multiple days. Each set of points represents the average and SEM of at least 10 parasites in one microscopic field of view. As noted in the text, in a given experiment all parasites in a microscopic field of view alkalinized with a similar rate but the rates varied significantly from trial to trial.